Azotobacter vinelandii is proposed to contain a single beta-ketothiolase activity participating in the formation of acetoacetyl-CoA, a precursor for poly-beta-hydroxybutyrate (PHB) synthesis, and in beta-oxidation (Manchak, J., Page, W.J., 1994. Control of polyhydroxyalkanoate synthesis in Azotobacter vinelandii strain UWD. Microbiology 140, 953-963). We designed a degenerate oligonucleotide from a highly conserved region among bacterial beta-ketothiolases and used it to identify bktA, a gene with a deduced protein product with a high similarity to beta-ketothiolases. Immediately downstream of bktA, we identified a gene called hbdH, which encodes a protein exhibiting similarity to beta-hydroxyacyl-CoA and beta-hydroxybutyryl-CoA dehydrogenases. Two regions with homology to bktA were also observed. One of these was cloned and allowed the identification of the phbA gene, encoding a second beta-ketothiolase. Strains EV132, EV133, and GM1 carrying bktA, hbdH and phbA mutations, respectively, as well as strain EG1 carrying both bktA and phbA mutations, were constructed. The hbdH mutation had no effect on beta-hydroxybutyryl-CoA dehydrogenase activity or on fatty acid assimilation. The bktA mutation had no effect on beta-ketothiolase activity, PHB synthesis or fatty acid assimilation, whereas the phbA mutation significantly reduced beta-ketothiolase activity and PHB accumulation, showing that this is the beta-ketothiolase involved in PHB biosynthesis. Strain EG1 was found to grow under beta-oxidation conditions and to possess beta-ketothiolase activity. Taken together, these results demonstrate the presence of three genes coding for beta-ketothiolases in A. vinelandii.